Summary Cell-cell interactions, mediated by adhesion and signaling receptors, are highly dynamic and subject to cytoskeletal movements that impart substantial mechanical force at the interface. How cells combine mechanical and biochemical signals to carry out specific functions is not well understood. Cells of the immune system present a compelling context for studying force transmission and mechanosensing because they are structurally dynamic and are sites of biochemical information transfer. T cell signaling is closely linked to the cytoskeleton, and it is evident that forces applied by the actin cytoskeleton at the T cell receptor are transduced to biochemical signaling leading to T cell activation. However, the molecular mechanisms by which these forces are regulated and how they contribute to T cell function remain obscure. In our parent award, we proposed to dissect the interactions and activities of proteins that reside at the intersection of actin and microtubule (MT) dynamics to advance our understanding of force generation and mechanosensing in T cells. We hypothesize that dynamic microtubules modulate the T cell cytoskeleton and proximal signaling both by 1) regulating actin polymerization dynamics in the lamellipodium and the assembly of structures in the lamella and 2) regulating RhoA activation leading to myosin contractility and force generation. Ultimately, we hypothesize that MT/actin interactions contribute to the ability of T cells to adapt their activation and effector function in response to the stiffness of target cells. Our preliminary studies have shown that there is considerable cross-talk between the actin and MT cytoskeletons. In our ongoing studies, we are examining mechanisms by which MT regulate actin dynamics by probing the specific interactions between MT and actin via +TIP proteins using optogenetic approaches. We are also dissecting the mechanisms that link dynamic MTs to myosin driven contractile force generation using traction force microscopy and optogenetic manipulation of myosin contractility. Finally, we will place our in vitro work in a functional context by testing our hypothesis that contractility tunes the mechanical coordination of cytotoxic T lymphocyte activation and their killing efficacy using primary cells. Simultaneous imaging of cytoskeletal dynamics and signaling combined with the measurement of the forces exerted by cells are key experiments for this project. This requires an imaging system capable of multicolor fluorescence imaging with high signal to noise ratio and low photo-toxicity. Spinning Disk Confocal Microscopy (SDCM) is an imaging technology that allows for visualization of molecular events deep within the cell interior and through optically clear substrates with high resolution perpendicular to the imaging plane. Furthermore, unlike scanning confocal microscopy, SDCM allows for rapid imaging (10s of Hz) with relatively low light levels and reduced photo-toxicity. Currently, the only SDCM instrument available to us is in a shared facility. However, this instrument has relatively low-power lasers, limiting the signal to noise ratios available when imaging T cells through 30-50 micron polyacrylamide gels to perform traction force microscopy (TFM). Moreover, the camera on this instrument has limited acquisition speeds and sensitivity (65% Quantum Efficiency) compared to the one on the microscope in our lab (95% QE, >100Hz frame rates). This supplement request is to add a spinning disk confocal unit to our current microscope. As TFM is directly related to all of our original Aims, this enhanced capability will inform all our proposed studies. Moreover, the high speed imaging capability will also enable us to perform super-resolution traction force microscopy using newly developed algorithms. Our studies of immune cell mechanosensing and the underlying pathways will advance our understanding of a number of immunodeficiencies and will help in providing new targets for intervention in immune therapy.